The Mechanism Of Reversal Of Multidrug Resistance In HCC Cells After Lipid Rafts Disruption By Cavl Knock-down And M?CD Treatment

Purpose:Two different treatments were used to disrupt the lipid rafts on the cell membrane of the SMMC7721/ADM cells:One was knocking down the Cav1(Cavl in short),which is one of the marker protein of lipid rafts,the other was depleting the cholesterol on the cell membrane by administration of methyl-?-cyclodextrin(M?CD).Verify if the multidrug resistance of the SMMC7721/ADM cells were reversed after lipid rafts disruption by MTS assay.The present study aimed to clarify the effects on the functions of two important ABC transporters P-gp and MRP 1,as well as the cell membrane permeability by these two different approaches.The mechanisms by which of each approaches reverse multidrug resistance are primarily explored,that is either mainly through mitigating active drug transport or the increase of passive drug diffusion also take a part.Methods:1.The SMMC7721/ADM cells were previously constructed.MTS assay was used to detect the proliferation inhibition rates of SMMC7721 parental strain and MDR strain under different concentrations of ADM incubation,and the half maximal inhibitory concentration(IC50)of the two cell lines and the drug resistance index(RI)of the MDR cell line were calculated.2.Three shRNA pairs were designed based on human Cavl gene to construct the SMMC7721/ADM-SiCavl cells.QPCR and Western Blotting were used to screen the cell lines with the best silencing effect for subsequent experiments.3.The proliferation inhibition rates of SMMC7721/ADM cells before and after Cavl knock-down and M?CD treatment were detected by MTS assay respectively,the half maximal inhibitory concentrations(IC 50)and reversal folds by different approaches were calculated.4.Flow cytometry was used to detect the changes of the mean fluorescence intensity of ADM in SMMC7721/ADM cells before and after M?CD treatment,with two different administration sequences of M?CD and ADM,so as to explore the the possibility that M?CD have an influence on cell membrane permeability.5.After 5ug/ml Rho123 incubation for 30min,the mean fluorescence intensity of Rho123 either in the presence or absence of LY335979 and MK571,in the SMMC7721/ADM cells before and after Cavl knock-down,as well as MpCD treatment were compared,respectively.The discrepancis of Rho123 accumulation between could reflect the passive diffusion of drugs.6.Cytotoxicity of M?CD was detected by LDH assay.7.According to the Rho 123 accumulation assay,the functional inhibition rates of P-gp and MRP1 of SMMC7721/ADM cells before and after Cavl knock-down,as well as M?CD treatment were calculated and compared pairwise to figure out the effect of Cavl knock-down and M?CD treatment on P-gp and MRP1 activities.8.MTS assay was used to detect the optical density of SMMC7721/ADM cells under 2,4,8ug/ml ADM incubation in the presence of L335979 and MK571.The survival rates of cells before and after Cavl knock-down,as well as M?CD treatment were compared respectively.Results:1.The proliferation inhibition rates of SMMC7721/ADM cells under 0.25ug/ml,lug/ml,4ug/ml,16ug/ml and 64ug/ml ADM were significantly lower than those of the SMMC7721/ADM parent strain.The half maximal inhibitory concentration(IC50)of the MDR strain and the parent strain was 4.75±0.3325ug/ml and 0.49±0.006ug/ml,respectivel y.2.The shRNA-Cavl plasmid was successfully constructed and validated correct by biosequencing.SMMC7721/ADM cells after interfering lentivirus transfection were screened by qPCR and Western Blotting.The silencing efficiency of SMMC7721/ADM-shRNA3 was the highest,reaching 90%.3.After Cavl knock-down and M?CD treatment,the proliferation inhibition rates of the SMMC7721/ADM cells under 0.25ug/ml,lug/ml,4ug/ml,16ug/ml and 64ug/ml ADM were all higher than control cells,IC 50 values were reduced to 2.703±0.094ug/ml and 2.173±0.094ug/ml,with the reversal fold of 1.68 and 2.19 respectively.4.In the case of M?CD treatment before ADM incubation,the mean fluorescence in SMMC7721/ADM cells after M?CD treatment was higher when detected upon ADM removal,and lower after 45min of ADM removal than before.In the case of ADM incubation before M?CD treatment,the mean fluorescence in SMMC7721/ADM cells after M?CD treatment was lower than before.5.Either in the presence or absence of LY335979 and MK571,the mean fluorescence intensity of Rho123 in SMMC7721/ADM cells after M?CD treatment was lower than before.In the absence of LY335979 and MK571,the mean fluorescence intensity in SMMC7721/ADM-SiCavl cells was about 20%higher than control.In the presence of LY335979 and MK571,the two are basically equal.6.The cytotoxicity rate of 5mM M?CD for 45min on SMMC7721/ADM cells was approximately 10%,and the cytotoxic effect of M?CD didn't show significant concentration or time dependence.7.The functional inhibition rates of P-gp and MRP1 in SMMC7721/ADM cells after Cavl knock-down and M?CD treatment were both lower than control by 20%,indicating that the two approaches of lipid rafts disruption could reduced the efflux functions of P-gp and MRP1 to some extent.8.In the presence of LY335979 and MK571,the optical density of SMMC7721/ADM cells after Cavl knock-down and M?CD treatment under 2,4,8ug/ml ADM were all lower than control.Conclusion:1.The multidrag resistance of SMMC7721/ADM cells could be reversed after lipid rafts disruption by means of Cavl knock-down and M?CD treatment.2.When M?CD depletes the cholesterol on the cell membrane,it also increases the cell membrane permeability,as well as the passive diffusion of drugs,which may lead to the reduction of multidrug resistance.3.After knocking down Cavl and M?CD treatment,the efflux activities of P-gp and MRP1 were weakened,which in turn led to the reduction of multidrug resistance to some degree.4.In addition to P-gp and MRP1,Cav1 knock-down may also affect the functions of other ABC transporters.Its influence on cell membrane permeability is negligible,so the multidrug resistance reversal is mainly through changing the active transport of drugs.